Quantitative microRNA Expression Analysis

Study personnel from the Mexico site were trained in the isolation and quantitation of microRNAs at the US site. Both sites employed the same study protocol for all assays. RNA was extracted from 200 μL of plasma using the miRNeasy serum/plasma kit (Qiagen). Purified RNA was converted to cDNA using the miScript II RT Kit (Qiagen) in 20 μL reaction volumes using the miScript HiSpec buffer. Real-time quantitative polymerase chain reaction (qPCR) was used to assess relative expression of candidate microRNAs using the miScript kit (Qiagen). Experiments were carried out using a 384-well (US) or 96-well (Mexico) plate format on a Bio-Rad CFX real-time PCR machine using the manufacturer’s recommended cycling conditions. A standard curve was constructed for each microRNA target using a series of five serial dilutions. Both sites obtained at least three replicates measures per sample for each microRNA target. MicroRNA expression levels were normalized using cel-miR-39 and the global geometric mean signal of all reliably detected microRNAs [20 (link), 21 (link)], and relative expression levels were calculated using the ΔΔCt method [22 (link)].